Abstract
Aim: The purpose of this study was to explore the parallel expression of platelet-derived growth factor receptor α (PDGFRα) and human epidermal growth factor receptor 2 (HER2) or p53 in relation to clinicopathological parameters of oral squamous cell carcinoma (OSCC) to define their role in progressive growth of tumor. Materials and Methods: Expression of PDGFRα, HER2 and p53 was evaluated in 71 OSCC samples by immunohistochemistry. HER2 status was verified by fluorescence in situ hybridization. Results: PDGFRα and p53 expression were associated with tumor grade (p=0.043 and p=0.040, respectively). HER2 expression was more frequent in advanced (III/IV) cancer (p=0.006). A positive correlation of PDGFRα with HER2 (r=0.267; p=0.024) and with p53 (r=0.266; p=0.025) was noted. PDGFRα/HER2 and PDGFRα/p53 co-expression was found more often in G3 than in G1 and G2 tumors (p=0.008 and p=0.015, respectively). Conclusion: Our study revealed that PDGFRα/HER2 and PDGFRα/p53 co-expression exists in poorly differentiated OSCCs, suggesting that cooperation between these proteins might enhance aggressive behavior of tumor.
Oral squamous cell carcinoma (OSCC) is among the 10 most common malignancies in the world and its incidence has escalated globally (1, 2). Despite recent advances in available therapeutic strategies, a high percentage of patients still have a poor response to therapy and high recurrence rates (2). The 5-year survival rate for OSCC patients has not improved and remains in the range of 50-60% (3).
The molecular mechanisms responsible for the development and progression of oral cancer are still poorly understood (3, 4). Genetic and molecular studies are limited by the heterogeneous histology, tumor site and biological diversity of oral tumors (3, 5). The pathomorphological features of oral carcinomas are often associated with overexpression/amplification of surface receptors and mutation/overexpression suppressor genes in tumor cells (4, 6). Therefore, an examination of the biological features of oral carcinomas with established clinical and pathomorphological parameters might be useful for screening subgroups of patients for individual therapy (7). Currently, among the biological factors, tyrosine kinase receptors [e.g. platelet-derived growth factor receptors (PDGFRs), human epidermal growth factor receptor 2 (HER2)] and p53 protein are being intensively investigated in solid tumors including OSCC (4, 5).
PDGFRα is a member of the transmembrane receptor tyrosine kinases family which plays a key role in the regulation of cell proliferation, chemotaxis and tumorigenesis by autocrine and paracrine stimulation (8, 9). PDGF is involved in angiogenesis in normal and tumor tissue (10). The dysfunction of PDGFs and their cognate receptors has been shown to play an important role in human carcinogenesis (11). Overexpression of PDGFR was associated with high malignancy of gliomas and advanced stage of head and neck carcinomas (10, 11). PGDFRs have been very rarely studied in OSCC and their role has not been established (9).
The second receptor belonging to the family of tyrosine receptors is HER2 (ERBB2). HER2 is a member of the epidermal growth factor receptor (EGFR) family involved in controlling cell growth and differentiation (5). In normal cells, the expression and activity of HER2 is strictly controlled (5). HER2 overexpression has been observed in many cancer types such as breast, gastric, colon, head and neck carcinoma, which are often associated with increased tumor size, tumor grade, metastasis activity and hence a poor prognosis (7, 12, 13).
The tumor suppressor gene TP53 plays critical role in repressing cancer invasion and metastatic progression. Recent evidence indicates that alteration in the TP53 gene represents common molecular changes in human cancer associated with a selective growth advantage and loss off of cell-cycle control (4, 14). The loss of p53 function with resultant overexpression has been reported in various human tumors such a breast, brain, rectum, colon, oesophagus, lung cancers and OSCCs (4). Previous studies have shown that oral carcinomas with overexpression/mutation of p53 grow faster and have aggressive nature and poor prognosis (1, 3, 15).
Up to now, co-expression of PDGFRα/HER2 and PDGFRα/p53 in oral carcinoma has not been examined and the relationship between these proteins which play different functions in tumor cells during tumor growth remains unclear. It seems to be important to analyze correlations between PDGFRα, which promotes angiogenesis of tumor, in relation with HER2 and p53 expression in OSCC.
The aim of this study was to evaluate parallel expression of PDGFRα and HER2 and p53 protein in OSCC in relation to clinicopathological parameters in order to define whether co-expression of these proteins determines the individual clinicopathological features of OSCCs and may be useful biomarkers characterizing their growth and behavior.
Materials and Methods
Patient tissues. A total 71 cases of excisional surgical specimens from 71 patients treated surgically for primary OSCC were obtained from the Department of Pathomorphology and Oncological Cytology of the Wroclaw Medical University, Poland between 2011-2014. Only the following tumor sites were included in the study: tongue, floor of the mouth, oral vestibule and hard palate. Patients with other localization of tumor, non-OSCC histopathology or treated before surgery were excluded. All tumors were histologically verified to confirm the diagnosis and categorize tumor grade, as well-, moderately or poorly differentiated according to the WHO classification (16). Clinical data for all 71 patients were analyzed to identify age, gender, tumor site and clinical stage. Clinical stage was classified in accordance with the 2009 American Joint Committee on Cancer staging criteria (seventh edition) (17). The distribution of clinical and histological features of the patients is shown in Table I. This study was approved by the Ethics Committee of Wroclaw Medical University (approval date 18th May 2016, no. KB-230/2016) and was performed in accordance with the declaration of Helsinki.
Immunohistochemistry (IHC). Immunohistochemical staining of PDGFRα, HER2, and p53 was performed on paraffin-embedded tissues from the selected blocks using the ABC method consisting of labeled streptavidin biotin reagents conjugated to peroxidase (LSAB+ Kit, HRP; Dako, Copenhagen, Denmark) and the following primary monoclonal antibodies: anti-PDGFRα (clone D13C6; Cell Signaling Technology, Danvers, MA, USA), anti-HER2 (clone CB11; Novocastra, Newcastle, United Kingdom) and anti-p53 (clone DO-7; Novocastra). All procedures were performed according to the manufacturer's protocols.
Five-micrometer sections from each selected block were deparaffinized and antigen-retrieved in citrate buffer (pH=6.0) by microwaving at 700W for three times for 5 minutes for each antibody. After microwave heating, the samples were cooled for 20 minutes. The activity of endogenous peroxidase was blocked by using 3% H2O2. Nonspecific binding of antibodies was blocked with 10% bovine serum albumin. Tissue preparations were treated with primary antibodies (anti-p53, dilution 1:50; anti-HER2, dilution 1:80; anti-PDGFRα, dilution 1:40) overnight at 4°C. After washing with 0.1 M Tris-buffer, pH 7.4 (TBS), the tissue samples were incubated (15 minutes at room temperature) with a secondary biotinylated antibody and with streptavidin-horseradish peroxidase-conjugated (EnVision detection Kit Peroxidase, DAB rabbit/mouse; Dako,). Following washing with TBS, the immunohistochemical reaction was detected by 3,3’-diaminobenzidine (Dako) as a chromogen (8 minutes at room temperature). Slides were then counterstained with hematoxylin. Internal positive controls were set as: astrocytoma for PDGFRα, breast carcinoma for HER2, colon carcinoma for p53. Negative controls were performed without primary antibody.
Clinicopathological characteristics of patients with oral squamous cell carcinoma.
Interpretation of IHC reaction. The slides were evaluated under an Olympus BX-51 double-headed light microscope (Olympus, Tokyo, Japan). The localization, distribution and intensity of PDGFRα, HER2 and p53 immunopositivity were evaluated in the tissue sections. Expression of PDGFRα and HER2 was assessed by determination of membranous immunostaining based on the intensity of immunostaining and the percentage of stained tumor tissue area. Immunostaining for PDGFRα and HER2 exceeding more than 10% of tumor cells was considered as positive. The percentage of p53-positive cells was determined by counting 1000 cells in 10 randomly selected high-power fields in relation to the total number of cells. Immunoreactivity was judged positive if immunostaining of p53 protein was observed in more than 10% of tumor cells. The intensity of immunoreaction was scored as: negative, weak, moderate, and strong. The immunostained slides were independently evaluated by two of the Authors who were blinded to clinical information of individual patients. The percentage of PDGFRα-, HER2- and p53-positive tumor cells were divided into four groups according to immunoreactivity and staining intensity as follows: negative: 0-10% positively stained cells, weakly positive: 11-30% positively stained cells, moderately positive: 31-50% positively stained tumor cells, strongly positive: more than 50% positively stained cells.
Association between clinicopathological parameters and protein expression in patients with oral squamous cell carcinoma.
Fluorescence in situ hybridization (FISH). FISH technique was performed on thin formalin-fixed paraffin-embedded tissue sections (n=17 cases with weak, moderate and strong HER2 immunoreactivity) using locus-specific identifier Pathvysion HER-2 Probe Kit (Vysis, Downers Grove, IL, USA). Spectrum Orange HER2 probe located at chromosome 17p11.2-q12 was used together with Spectrum Green Dual Colour probe located at the centromere of chromosome 17 (17p11.1-q11.1) according to the methodological procedure of Abbott/Vysis. Sections were deparaffinized in xylene (3×5 minutes) and dehydrated in 99.8% ethanol (3×1 minute). Tissue samples were pre-treated by immersing in 0.2 N hydrochloric acid for 20 minutes to avoid tissue autofluorescence, then the slides were rinsed in purified water for 5 minutes and washed twice in standard saline citrate (SSC) for 5 minutes. The slides were then heated at 80°C in sodium thiocyanate solution for 30 minutes, followed by rinsing for 1 minute in distilled water and washing in 2×SSC. The sections were then subjected to protease at 37°C for 35 minutes, washed in 2×SSC for 5 minutes and air-dried. After that HER2 probe added to each slide which was then coverslipped. Samples were denatured for 5 minutes at 72°C. After that hybridization was performed for at least 16 hours at 37°C in DAKO Hybridizer™ equipment. Then preparations were washed with 2×SSC at room temperature. After coverslips were removed, slides were immersed in 2×SSC/0.3% NP40 at 72°C. After air-drying in the dark, slides were counterstained with 4’6’-diamino-2-phenylindole (DAPI). The slides were examined using an Olympus BX-61 fluorescence microscope (Olympus, Tokyo, Japan) with filters suitable for fluorescein/rhodamine and DAPI. Images were analyzed using CellF Imaging Software (Olympus, Tokyo, Japan). The methodology was performed according to ISO/IEC 17025:2005 accreditation. HER2 gene amplification was considered as positive when the FISH ratio was higher than 2.2 or HER2 gene copy was more than 6.0. In cases with polymorphism of HER2 gene, polysomy of chromosome 17 was excluded by TP53 gene analysis using Vysis LSI TP53 (17p13.1) Spectrum Orange Probe (Vysis, Downers Grove, IL, USA) (18).
Membranous expression of platelet-derived growth factor receptor α (PDGFRα) (A) and human epidermal growth factor receptor 2 (HER2) (B) in cancer cells. Strong nuclear expression of p53 protein in cancer cells (C) (EnVision technique). Scale bar=50 μm.
Statistical analysis. Correlations between PDGFRα, HER2, p53 expression and clinicopathological parameters were statistically studied by chi-square test or nonparametric tests. Associations between proteins were analyzed by Spearman's rank correlation. Differences were considered as statistically significant when p<0.05. Statistical tests were performed using STATISTICA v12.0 (StatSoft, Krakow, Poland).
Representative case of oral squamous cell carcinoma analyzed for human epidermal growth factor receptor 2 (HER2) gene status by fluorescence in situ hybridization technique. Positive (A) and negative (B) for HER2 gene polymorphism in oral squamous cell carcinoma, Green: Centromere region; orange: HER2 gene. Magnification ×600.
Results
The presence of PDGFRα was observed on the membrane of tumor cells in 26/71 (36.6%) cases. Positivity for PDGFRα was found at various levels in tumor tissue (11-70%), but most positive cases (80.8%) presented weak intensity and immunoreactivity (Figure 1A). In some cases of OSCC, PDGFRα expression was observed in both tumor and stromal cells. PDGFRα expression was seen more frequently in grade (G) 3 tumors compared to G1 and G2 tumors (p=0.043). There were no statistical correlations noted between PDGFRα expression and other clinicopathological parameters (Table II).
Membranous HER2 immunostaining was noted in 55 out of 71 (77.5%) cases and ranged from 11 to 90% of tumor tissue (Figure 1B). Most OSCCs showed strong intensity and high immunoreactivity for HER2 in more than 50% of tumor tissue (43.6% of all positive cases). It was revealed that HER2 expression was not comparable with HER2 gene status: HER2 gene polymorphism was present in 2/17 (11.8%) cases (Figure 2). We found that HER2 immunoreactivity was more frequent in advanced (III/IV) clinical stage of disease as compared with low (I/II) stage (p=0.006). No correlations between HER2 expression and other clinicopathological parameters were observed (Table II).
Spearman's rank correlation between expression levels of studied proteins in oral squamous cell carcinomas.
Expression of p53 was found in 33/71 (46.5%) tumors. Nuclear accumulation was observed in different ranges from 11 to 90% tumor tissue (Figure 1C). Strong intensity and immunoreactivity was observed in 33.3% of all positive cases and dominated in glandular structure of tissue. No positive reaction was observed in stroma cells. P53 expression was associated with patient age (p=0.034). The presence of p53 was significantly more often in poorly differentiated carcinomas (G3) than in moderately (G2) and well-differentiated tumors (G1) (p=0.040). There were no statistical correlations between p53 expression and other clinicopathological features (Table II).
Interestingly, we found positive correlation of expression of PDGFRα with HER2 (r=0.267, p=0.024) and with p53 (r=0.266, p=0.025) expression by IHC in the group of OSCCs analyzed (Table III). To determine the association between co-expression of PDGFRα/HER2 and PDGFRα/p53 in relation to clinicopathological parameters of OSCCs, the total group of oral carcinomas was subdivided into two subgroups according to co-expression-positive and co-expression-negative cases. Further analysis between co-expression of studied biomarkers and clinicopathological parameters of OSCCs was performed separately by subgroup and compared. As shown in Figure 3, the co-expression of PDGFRα/HER2 and PDGFRα/p53 was found more often in poorly (G3) than in well- (G1) and moderately (G2) differentiated tumors; the observed differences were statistically significant (p=0.008 and p=0.015, respectively).
Correlation between protein immunophenotypes and clinicopathological parameters of oral squamous cell carcinomas. High tumor grade was associated with platelet-derived growth factor receptor α/human epidermal growth factor receptor 2 (PDGFRα+/HER2+) immunophenotype (p=0.008) (A), and with PDGFRα+/p53+ immunophenotype (p=0.015) (B).
Discussion
In the present study, we analyzed co-expression of PDGFRα with HER2 or p53 in OSCC samples to investigate the role of these molecules in progressive growth of tumor and understanding of mechanisms responsible for the aggressive nature of OSCC. To our best knowledge, this is the first report describing co-expression of PDGFRα with HER2 or p53 in relation to clinicopathological parameters of OSCC.
PDGFRα immunohistochemical expression in OSCC has been analyzed very rarely and its role in tumor growth is still controversial. Several authors indicated that autocrine and paracrine stimulation of PDGFRα could play a crucial role in oral and head and neck tumorigenesis and progression (8-10). One study showed that during tumor growth, PDGFRα stimulation can initiate the activation of different signaling receptors, e.g. EGFR and Notch, which leads to dedifferentiation of tumor cells, and increased cell motility and aggressiveness of tumor (11). In the present study, PDGFRα expression observed in some sets of oral carcinomas might increase activation of signaling pathways in tumor cells which depend on this receptor (8). We might suggest that oral carcinoma cells with higher PDGFRα expression might present more aggressive behavior (11). Our suggestion is supported by the association revealed between PDGFRα expression and high grade of OSCC found in the current study which may be partly comparable to other results which suggest that this receptor is a crucial factor in the regulation of angiogenesis (8, 11). Some authors postulated that PDGFRα might indirectly facilitate spread of carcinoma cells from primary tumor tissue and invasion of surrounding normal tissue (8, 9). In our opinion, the association between PDGFRα expression and poorly differentiated tumor cells might have influence cell motility and facilitate their migration through the basement membrane. Similarly to liver and breast carcinoma, high PDGFRα expression in OSSC might contribute to reduced cell–cell adhesion and increased spread of carcinoma cells (19). In vitro experimental study revealed that suppression of PDGFRα/β tyrosine phosphorylation inhibits carcinoma cell migration (10). This confirms the essential role of PDGFRα in tumor cell invasion (10).
Early reports of HER2 overexpression in head and neck squamous cell carcinomas range from 0-47% (20). However, in laryngeal cancer, positive HER2 immunostaining was found in 68.0% of cases (21). In our study, IHC staining of HER2 was observed in 77.5% of OSCC. These results are similar to those of Dalal et al. (13), who detected HER2 positivity in 62.2% of OSCC. Moreover, many published results showed HER2 expression to be rare in OSCC (5, 12, 22). Controversial results in different studies might be due to divergent patient cohorts (location of lesions, gender) or differences in IHC methods, type of antibody and tissue preparation (5, 13). Similarly to earlier reports (5, 12, 13), we recorded HER2 gene polymorphism in small number of cases. A high concordance between HER2 by IHC and FISH observed by other authors (5, 12) was not found in our study. Most likely, our contrary results may be due to methodical differences and interpretation of IHC staining (5). In oral carcinoma, validation of HER2 IHC and FISH was not performed (5). There are no standardized criteria for IHC and FISH evaluation of HER2 in OSCC (5, 12). Similarly to previous data our results revealed that clinical implication of HER2 expression/polymorphism in OSCC is limited (5, 12, 23). Similarly to other reports (5, 24), we did not find a significant relationship between patient age and HER2 overexpression.
P53 expression was found in 46.5% of samples, which is consistent with previous studies (1, 3, 25). The detection of nuclear p53 accumulation in a large percentage of OSCC in our study indicates that p53 alteration is a frequent event in OSCC (3, 4). Moreover, it is suggested that nuclear accumulation of p53 protein in an inactive form might play a key role in tumorigenesis and progression of OSCC (3). Therefore, it seems reasonable to assume that high p53 protein expression levels detected by IHC reflect the extended half-life of p53 protein encoded by TP53 gene mutation (3). Similarly to published studies (3, 14, 15) we found correlation between p53 overexpression and grade of OSCC. The authors postulated that patients with OSCC whose tumor showed high expression of p53 protein had worse clinicopathological parameters and presented aggressive behavior (3, 14). In contrary to earlier data (26), we found p53 expression more frequently in tumor of patients over 62 years old than those younger. Results from the current study indicate that in older patients with OSCC, the risk of p53 alteration is higher and may be the result of accumulation of p53 DNA damage. No significant differences between p53 expression according to clinical tumor stage were observed in the present study, indicating that p53 alteration might be early event in oral carcinoma growth (4, 26, 27). However, there are data (3, 14) of p53 protein expression being more frequent in advanced clinical stage of tumor.
The current study is the first that revealed the co-expression of PDGFRα with HER2 as well as with p53 protein in oral carcinomas. There are no data as far as we are aware analyzing the correlation between these proteins in oral cancer but results of this study might be partly comparable with other studies (3, 14, 26, 27) that found positive correlation between p53 and other proteins such as inducible nitric oxide synthase (iNOS), homeobox transcription factor Nanog (NANOG), E-cadherin, and Kangai 1 (KAI-1) in oral cancer. These studies showed that parallel expression of analyzed proteins may exert a synergistic effect on the development, invasion and metastasis of oral carcinoma (3, 14, 26, 27). Recently, Kurahara et al. found a correlation between overexpression of p53 protein and PDGFRβ expression in pancreatic cancer. The authors reported that mutant p53 protein overexpression induced PDGFRβ expression, leading to enhancement of invasion of pancreatic cancer (28). In the present study, both PDGFRα and p53 protein expression were correlated. Based on the study of Kurahara et al. (28), we might suggest that similar cooperation between PDGFRα and p53 protein exists in oral carcinoma. Relevant to a previous study (29), the positive correlation between PDGFRα and HER2 expression found in this study might suggest that parallel activation of these receptors belonging to the same family is possible, thereby a paracrine mechanism of activation of co-expressed receptors might be linked to tumor progression. Interestingly, we found that in poorly differentiated oral carcinomas p53 and HER2 expression positively correlated with PDGFRα expression. Based on earlier published data (3, 14, 26, 27), and our observations, we may suggest that co-expression of PDGFRα/HER2 or PDGFRα/p53 might be a biological feature characterizing a subset of oral carcinomas showing tumor aggressive growth. On the other hand, observed co-expression of PDGFRα/HER2 and PDGFRα/p53 in poorly differentiated OSCC indicate these molecules combined with morphology of tumor cells might characterize a more invasive phenotype of OSCC.
In conclusion, the findings from this study suggest that cooperation between PDGFRα and HER2 or p53 proteins might lead to more aggressive behavior of OSCC. We propose that co-expression of PDGRFα/HER2 or PDGRFα/p53 in poorly differentiated OSCC indicates that these proteins may cooperate in invasion and enhance progression of OSCC.
Acknowledgements
The Authors offer special thanks to Dr Piotr Grelewski for his excellent technical assistance. This study was supported by Wroclaw Medical University (grant no. ST.B132.16.052).
Footnotes
Conflicts of Interests
None to declare.
- Received November 3, 2017.
- Revision received November 25, 2017.
- Accepted November 28, 2017.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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